We are testing the hypothesis that dynamin acts as a mechanochemical enzyme that undergoes a conformational change upon GTP hydrolysis producing membrane fission, a mechanism that we predict is common among the dynamin family members. Dynamin is necessary for internalizing essential nutrients, synaptic vesicle recycling and is tightly coupled to cell signaling events. More recently, dynamin has been linked to the peripheral neuropathy, Charcot-Marie-Tooth disease (PH domain mutation) and to a centronuclear myopathy (CNM, middle domain mutation). Examining the structure of dynamin in different conformations will help elucidate a mechanism of action for membrane constriction and fission, and provide insight into dynamin associated diseases. The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and, more recently, has been associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. Over the years, our structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the first three-dimensional structure of dynamin. All evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. The tension created by dynamin constricting the neck of coated pits may be sufficient for membrane fission in the cell. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. Previously, we solved the structure of a dynamin mutant (lacking its C-terminus) in the constricted and non-constricted states using using helical reconstruction and the IHRSR methods. The 3D volumes reveal three distinct radial densities, outer, middle and inner layers. During constriction the most obvious change is a decrease in the axial repeat and radius. However, the volume interiors shows a large conformational change within the middle layer, which provides a clue to the mechanism of constriction. Dynamin contains five identifiable domains: GTPase, middle, pleckstrin homology (PH), GTPase effector (GED) and proline/arginine-rich (PRD). Using molecular modeling tools, we docked the crystal structures of the GTPase and PH domains of dynamin into the 3D maps using a rigid-body Monte Carlo algorithm. The GTPase domain docked into the outer radial density while the PH domain docks into the inner radial density. The GED would then reside in the middle layer, which fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The results show how adjacent GTPase structures associate with one another in an arrangement consistent with the cryo-EM structure and suggest a mechanism for self-assembly and corkscrew motion during constriction. The positioning of the PH domain within the inner radial density places the variable loops facing toward the membrane. This positioning is consistent with the Charcot-Marie-Tooth mutation in the PH domain having an effect on lipid binding. Recently, we have solved the structure of full-length dynamin in the constricted and non-constricted states. To solve the structure of dynamin in the constricted state we used a dynamin mutant, K44A. The 3D reconstruction of full-length dynamin compared to the previous maps solved without the C-terminal PRD region, showed that the C-terminal PRD significantly changed the packing of the dynamin dimer in the dynamin-lipid tube. In addition, we have used immunogold labeling to confirm the location of dynamin domains within the dynamin tubes. In agreement with the molecular modeling, antibodies against the GTPase domain decorate the outer surface of the dynamin tubes. Antibodies against the C-terminus also decorated the outer surface suggesting the PRD is surface exposed and accessible for binding to several of dynamin partners. The 3D map also revealed that in the presence of GTP, K44A-dynamin constricts the lipid bilayer to an inner diameter of 2 nm. This is approaching the minimal requirement necessary for spontaneous membrane fission.